Within our continued effort aimed at developing conjugate vaccines for infectious diseases from synthetic fragments of bacterial polysaccharides we have three ongoing projects. Two are concerned with a vaccine for cholera and one with a vaccine for anthrax. Existing vaccines for these diseases are based on cellular material and, as such, in addition to having undesirable side effects, do not provide long-term immunity. Development of such vaccines is important from both the point of view of public health and of national interest. Development of a potent vaccine for cholera is important because of the involvement of our military in protecting US interests in developing third world countries. While anthrax does not constitute a major health problem in the civilized world, new concerns regarding anthrax have emerged because of potential use of some form of Bacillus anthracis, the etiological cause of anthrax, as a biological weapon. Our work towards a potent conjugate vaccine for cholera involves synthesis of oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugation of these antigens to suitable carriers, and serological evaluation of the immunogenicity of the resulting neoglycoconjugates. The approach towards a vaccine for anthrax is based on preparation of a neoglycoconjugate from a suitable carrier and the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium. In the cholera project, we have simplified and made more efficient the synthesis of the hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae O1 using glycosyl donors and glycosyl acceptors having the N-(3-deoxy-L-glycero-tetronamido) side chain already in place. In addition, we have investigated the role of architectonic details in the neoglycoconjugates upon immunogenicity. We have previously evaluated serological responses of conjugates from the hexasaccharide that mimics the O-PS of Vibrio cholerae O:1. Those conjugates were found potent immunogens, and, in mice, they also elicited bactericidal and protective antibodies for Vibrio cholerae O:1. To approximate a clinically relevant vaccine, we prepared conjugates composed of BSA as a carrier and synthetic hexasaccharide of the O-specific polysaccharide of V. cholerae O1, serotype Ogawa (CHO) equipped with different length linkers, and have evaluated the influence of the linker length upon immunogenicity. The length of time between booster immunization was also examined. Evaluated also was immunogenicity of conjugates made from Ogawa mono- and tetrasaccharide with that made from the hexasaccharide. The immunogenicity of the synthetic immunogens was compared with that of Ogawa LPS. The Ogawa LPS and Ogawa neoglycoconjugates were delivered intraperitoneally, and were therefore likely to have targeted the same B cell populations. LPS or the CHOBSA neoglycoconjugates delivered separately resulted in similar humoral responses but the efficacy of the antisera were qualitatively different. The intriguing observation was that priming B cells with suboptimal doses of native LPS, followed by a boost with the neoglycoconjugates of various saccharide lengths, led to a very efficient production of vibriocidal antibodies. The former strategy may be particularly effective in young children who were previously exposed to or would be recovering from cholera, who routinely require multiple exposures to an antigen before they would be considered immune. Serological evaluation of antibodies resulting from immunization of mice with conjugates incorporating various length of linker showed that antibodies given rise to by the antigen equipped with the longest linker were least efficient in rendering protection. In addition, in order to determine whether immune response may be elicited against V. cholerae LPS using transcutaneous immunization (TCI), we immunized mice with a conjugate made from the Ogawa hexasaccharide and BSA. Our results suggest that TCA with the conjugate is safe and immunogenic, resulting in prominent anti-V. cholerae serum IgG responses, and warrants further evaluation. Regarding our work towards a vaccine for cholera caused by the strain Vibrio cholerae O:139, we have prepared the conjugation ready, spacer-equipped, phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide. [unreadable] Within the anthrax project, we have synthesized the linker-equipped tetrasaccharide and all theoretically possible fragments thereof. The compounds were instrumental in developing an efficient test (photogenerated glycan array) for presence of anthrax spores.[unreadable] We also continue our international collaboration with Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic, on mass spectroscopy of carbohydrates that are involved as tools in the life sciences. In this line of work we have investigated negative matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry fragmentation of synthetic analogs of the O-specific polysaccharide of Vibrio cholerae O:1.